Optical arrangement for the production of a light-sheet

ABSTRACT

The invention is directed to an optical arrangement with a light source for emitting a light bundle and with optical elements for transforming this light bundle into the shape of a light sheet, particularly suitable for illuminating individual planes of a three-dimensional specimen in selective plane illumination microscopy (SPIM). According to the invention, means are provided for varying the cross section of the light sheet, for varying the length of the light sheet and/or for influencing the direction in which individual beam components extending within the light sheet are directed to the specimen substance. This makes it possible to adapt the geometry of the light sheet to the illumination requirements for observing one and the same specimen plane with a plurality of different objectives and, if required, to reduce shadows occurring within the observed specimen plane as a result of the illumination.

CROSS-REFERENCE TO RELATED APPLICATION

Notice: More than one reissue application has been filed for the reissueof U.S. Pat. No. 7,787,179. The reissue applications are U.S. patentapplication Ser. No. 13/600,663 (now abandoned), filed on Aug. 31, 2012,which is a reissue patent application of U.S. Pat. No. 7,787,179, filedas U.S. patent application Ser. No. 11/946,232 on Nov. 28, 2007; andU.S. patent application Ser. No. 13/932,475 (the present application),filed on Jul. 1, 2013, which is a divisional reissue patent applicationof U.S. patent application Ser. No. 13/600,663.

This application claims priority of German Application No. 10 2007 01508.8, filed Mar. 29, 2007, the complete disclosure of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to an optical arrangement with a light sourcefor emitting a light bundle and with optical elements for transformingthis light bundle into the shape of a light sheet, particularly suitablefor illuminating individual planes of a three-dimensional specimen inselective plane illumination microscopy (SPIM).

b) Description of the Related Art

In contrast to confocal laser scanning microscopy (LSM), in which athree-dimensional specimen is scanned point by point in individualplanes of different depth and the image information thus acquired isthen combined to form a three-dimensional image of the specimen, theSPIM technique relies on widefield microscopy and makes it possible todisplay an image of the specimen based on optical sections throughindividual planes of the specimen.

The advantages of the SPIM technique include the greater speed at whichimage information is acquired, the reduced risk of bleaching biologicalspecimens, and an expanded penetration depth of the focus in thespecimen.

In principle, in the SPIM technique fluorophores which are by themselvescontained in the specimen or have been introduced into the specimen forcontrasting are excited by laser light, and the laser radiation isformed into a light sheet, as it is called. A selected plane in thedepth of the specimen is illuminated by the light sheet and an image ofthis specimen plane is acquired in the form of an optical section byimaging optics.

To illustrate the geometry of the light sheet more clearly, it will beassumed in the context of the present invention that the light sheet hasa cross section which extends in the X and Y coordinate directionsperpendicular to the beam direction of the laser light and a lengthwhich extends in the Z coordinate direction along the beam direction.

The optical axis of the objective by which the illuminated specimenplane is to be imaged or observed is oriented perpendicular to the Zcoordinate direction.

Optical arrangements for generating a light sheet in connection with theSPIM technique are described in DE 102 57 423 A1 and DE 10 2005 027077A1.

These arrangements produce only a rigid light sheet which is notvariable with respect to its thickness, which should correspond to theextension in the X coordinate. This circumstance is particularlydisadvantageous when one and the same plane of a specimen is to beimaged successively in time with different objectives whose opticalimaging characteristics differ from one another.

In such cases, it is desirable to have the possibility of adapting thegeometry of the light sheet, above all, its thickness, to the respectiveobjective so that only the plane of interest is actually illuminated inthe specimen and, accordingly, an unwanted bleaching of the specimensubstance outside this plane is prevented. Also, the depth of focus ofthe light sheet can be adapted in this way to the respective objectfield being observed.

Another disadvantage in the arrangements mentioned above is that thelight sheet that is generated with them has a Gaussian intensity profilein cross section so that a uniform illumination of the specimen planebeing examined is impossible.

WO 2004/0530558 A1 describes a method in which a light-sheet typeillumination is generated based on a relative movement between aline-shaped light field and the specimen to be observed. The light-sheettype illumination is carried out in that the light field is lined upmultiple times successively in time due to the relative movement. Thishas the disadvantage that shadows result within the plane of thespecimen being examined because of portions of the specimen substancewhich are not transparent for the illumination light and which lie inthe illumination direction.

EP 0 248 204 B1 describes the generation of a line-shaped illuminationwith a linear fiber array and cylindrical lenses arranged downstream.However, again, the geometry of the light sheet cannot be varied.

The publication U.S. Pat. No. 4,826,299 describes the shaping of a lightsheet with a Powell lens. The Powell lens has an aspherical profile inone coordinate direction and is flat in the coordinate orthogonal to itso that a virtually homogenized line-shaped light field is formed from alight bundle and can be used as a light sheet. However, again thegeometry of this light sheet can not be varied, specifically withrespect to its thickness and length, so that an optimal illuminationthrough the specimen plane to be examined is also impossible in thiscase when using different objectives whose optical characteristicsdiffer from one another.

All of the arrangements mentioned above have the disadvantage ofunwanted shadows within the plane of the specimen being examined whichare caused by portions of the specimen substance in the illuminationdirection which are not transparent for the illumination light.

OBJECT AND SUMMARY OF THE INVENTION

Proceeding from this prior art, it is the primary object of theinvention to provide an optical arrangement for generating a light sheetwhich makes it possible to observe individual planes of a specimen witha greater efficiency than in previously known arrangements.

According to the invention, in an optical arrangement for generating alight sheet of the type mentioned above, means are provided for varyingthe cross section of the light sheet, for varying the length of thelight sheet and/or for influencing the direction in which individualbeam components extending within the light sheet are directed to thespecimen substance.

This makes it possible to adapt the geometry of the light sheet to theillumination requirements for observing one and the same specimen planewith a plurality of different objectives and, if required, to achieve areduction in shadows occurring within the observed specimen plane as aresult of the illumination.

In a first construction of the arrangement according to the invention, alight source is provided which emits a bundle of coherent light in whosepath are provided:

-   -   a collimator,    -   an aspherical optical element,    -   a lens or lens group for realizing a field diaphragm plane, and    -   a lens or lens group for realizing an aperture diaphragm plane.

In this connection, a bundle of parallel light is initially generated bythe collimator and is then transformed into the shape of the light sheetby means of the aspherical optical element. A Powell lens, for example,can be used as an aspherical optical element.

The cross section of the light sheet is adapted to the illuminationrequirements of a particular objective with a field diaphragm which isplaced in the field diaphragm plane and whose diaphragm opening isadequate for the desired cross section. If the objective is exchangedfor an objective with a different aperture or different imaging scale,for example, the field diaphragm is exchanged for a field diaphragmwhose diaphragm opening corresponds to the desired cross section. Byexchanging the diaphragms, a change in cross section with respect to thewidth of the light sheet is achieved, that is, its extension in the Ycoordinate is varied.

The diaphragms can be arranged on a changer wheel, for example. Changingthe diaphragms can be carried out manually or automatically, and therespective field diaphragm is chosen depending on the characteristics ofthe objective that is used.

A similar procedure is followed with the aperture diaphragms which areplaced in the aperture diaphragm plane depending on the characteristicsof the objective that is used. Exchanging the aperture diaphragmsinfluences the geometry of the light sheet with respect to its thicknessand its length. The reason that the thickness and length are influencedconjointly is that the depth of focus range depends upon the inversesquare of the numerical aperture when the light sheet is generatedanamorphotically by the aspherical optical element.

In an alternative construction, the influencing of the cross section isnot carried out, as was described, by exchanging diaphragms, but byplacing one or two zoom optics in the illumination beam path.

For this purpose, the lens group for realizing a field diaphragm and/orthe lens group for realizing an aperture diaphragm are/is designed insuch a way that they have a variable focal length.

Further, a device for reducing shadows in the specimen is provided inthis first embodiment of the arrangement according to the invention.

To this end, a wobble plate is arranged in a pupil plane of the beampath which has already been shaped to form the light sheet, or anoscillating mirror or a polygon scanner is positioned in a planeconjugate to the field diaphragm plane.

Owing to the deflecting movement generated in this way, the direction ofthe beam components of the light sheet is influenced in such a way thatthese beam components strike the specimen substance successively in timein alternating directions so that shadows caused by opaque specimensubstances within the illuminated specimen plane are prevented or atleast substantially reduced.

In a second construction of the arrangement according to the invention,a light source is provided which emits a bundle of spatially partiallycoherent light, and one or two cylindrical-lens arrays and fixed orvariable collimating optics are arranged in the path of this lightbundle, the lateral coherence length being less than the period of thecylindrical-lens arrays.

The cylindrical-lens arrays take over the function of a honeycombcondenser in a plane, for example, the Y-Z plane, wherein the individualbeam components or partial apertures generated by the first array arespatially superimposed in the field diaphragm plane with or without thehelp of a second array and the collimating optics. When a second arrayis present, the intensity distribution in the field diaphragm plane ismore homogeneous than when a second array is not present. The geometryof the light sheet can be adapted by means of variable collimatingoptics.

The spatially partially coherent light is generated, for example, bymeans of a temporally partially coherent light source such as abroadband laser, a dispersive optical element being placed in its beampath for purposes of reducing the spatial coherence. A grating, a prismor a stepped mirror can be used as a dispersive optical element.

In a third construction of the arrangement according to the invention,the light source comprises an array of individual laser light sources.Located downstream of these light sources are a cylindrical lens or GRINlens extending over the entire array and collimating optics. Every laserlight source completely illuminates the entire desired light sheet. Thewidth of the light sheet in the Y-Z plane is determined by the emittedaperture of the laser light sources and the focal length of thecollimating optics. The thickness of the light sheet in the X-Z plane isdetermined by the emitted aperture of the light sources and the focallengths of the cylindrical optics and collimating optics. Because of thespatial arrangement of the laser light sources, every light sourceilluminates the specimen plane at a different angle so that shadowswithin the specimen plane are prevented or at least substantiallyreduced as was described above.

With GRIN lenses, in contrast to conventional collector lenses, thefocal length is influenced by a continuous variation of the refractiveindex in the lens material.

In a fourth construction of the arrangement according to the invention,a micro-optical element for transforming the light bundle into the shapeof a light sheet is arranged in the light bundle coming from a coherentlight source and exiting from a light-conducting fiber.

This micro-optical element can be micro-optics provided with opticallyactive free-form surfaces or micro-optics which are constructed in theform of a GRIN lens and which have simultaneously a homogenizing effectin one cross-sectional direction and a focusing effect in the orthogonalcross-sectional direction.

A fifth construction of the arrangement according to the inventionprovides a light source which emits a bundle of coherent light withinwhose path the following components are provided:

-   -   anamorphotic optics, for example, in the form of a cylindrical        lens telescope, for generating a light bundle with an elliptic        cross section,    -   an optical deflection device, for example, in the form of an        oscillating mirror, a polygon scanner or a digital micromirror        device (DMD) for generating a scanning movement of this light        bundle, and    -   a focusing lens or lens group through which the scanned light        bundle is directed to the specimen.

In a manner known per se, the DMD comprises about 500,000microscopically small mirrors which can be tilted very quicklyindividually.

A light-sheet-like illumination is generated by this arrangement in thatthe light bundle which has an elliptic cross section and is focused inthe specimen is lined up multiple times successively with respect totime due to the scanning movement. The lining up of the individuallyfocused light bundles yields the geometry of the light sheet.

Shadows due to non-transparent specimen substances within theilluminated specimen plane are prevented or reduced in this constructionof the arrangement according to the invention by the cylindrical lenstelescope through which the direction of individual beam components isinfluenced as was described above.

The invention will be described more fully in the following withreference to embodiment examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 shows the principle of a first construction of the arrangementaccording to the invention with diaphragms for influencing the geometryof the light sheet;

FIG. 2 shows the principle of the first construction of the arrangementaccording to the invention, but in this case with zoom optics forinfluencing the geometry of the light sheet;

FIG. 3 shows the principle of the first construction of the arrangementaccording to the invention with a wobble plate for reducing shadows;

FIG. 4 shows the principle of the first construction of the arrangementaccording to the invention, but with an oscillating mirror for reducingshadows;

FIG. 5 shows the principle of a second construction of the arrangementaccording to the invention with cylindrical-lens arrays for shaping thelight sheet and for reducing shadows;

FIG. 6 shows an example for generating spatially partially coherentlight for realizing the construction according to FIG. 5;

FIG. 7 shows the principle of a third construction of the arrangementaccording to the invention with an array of individual laser lightsources for shaping the light sheet;

FIG. 8 shows the principle of a fourth construction of the arrangementaccording to the invention in which the light sheet is shaped by amicro-optical element;

FIG. 9 shows the principle of a fifth construction of the arrangementaccording to the invention in which the light sheet is shaped byone-dimensional scanning of a light bundle; and

FIG. 10 shows a first illustration of the prior art.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In FIG. 10, which will serve in the following for a brief explanation ofthe principle of selective plane illumination microscopy (SPIM) in theprior art, a specimen 1, for example, a biological substance, issurrounded by a transparent gel 2. This specimen 1 is athree-dimensional specimen 1 which extends in the drawing plane andperpendicular to the drawing plane. It will be assumed that the gel 2 isshaped to form a circular cylinder with an axis of rotation orientedperpendicular to the drawing plane.

As was already slated above, three-dimensional images of the specimen 1are obtained on the basis of a plurality of optical sections throughindividual specimen planes by the SPIM technique. It will be assumedthat the plane of the specimen 1 to be observed has the thickness d inthe drawing plane.

In order to observe this plane, a light sheet 3 having a length 1 and athickness d in the drawing plane which remains as uniform as possiblealong the entire length 1 and which extends

perpendicular to the drawing plane at least over the entire dimensioningof the specimen 1 is required.

In the context of the present description of the invention, it will beassumed that the length 1 of the light sheet 3 extends in the Zcoordinate direction, its thickness d extends in the X coordinatedirection, and its width b extends perpendicular to the drawing plane inthe Y coordinate direction. The Z coordinate direction also correspondsto the direction of the illumination beam path.

FIG. 1 shows a first construction of the arrangement according to theinvention. A light source (not shown) emits a coherent light which exitsfrom a light-conducting fiber 4 as a light bundle 5 and is directedthrough a collimator 6 to an aspherical element 7.

By passing through the aspherical element 7, the light bundle 5 isexpanded in the Y-Z plane shown in FIG. 1a, while the light bundle 5passes the aspherical element 7 in the X-Z plane, shown in FIG. 1b,substantially unchanged.

This reshaping yields the thickness d of the light sheet 3 in the Xcoordinate direction and its width b in the Y coordinate direction.

As is further shown in FIG. 1a and FIG. 1b, the illumination beam pathwhich is shaped to form the light sheet 3 first passes a lens group 8,for example, an achromat, to realize a field diaphragm plane 9, thenanother lens group 10 to realize an aperture diaphragm plane 11 and,finally, a third lens group 12 constructed as focusing optics throughwhich the light sheet 3 is directed into the specimen 1.

When a field diaphragm 13 is placed in the field diaphragm plane 9, thegeometry of the light sheet 3 is defined with respect to its width b bythe diaphragm opening of the field diaphragm 13. The diaphragm openingof an aperture diaphragm 14 placed in the aperture diaphragm plane 11determines the thickness d and the length 1 of the light sheet 3.

In this way, with a field diaphragm 13 of a determined diaphragm openingand an aperture diaphragm 14 of a determined diaphragm opening, thecross section and the length 1 of the light sheet 3 can be adapted to anobjective used to observe the selected plane of the specimen 1.

If this objective is exchanged for a different objective to observe thesame plane of the specimen 1, for example, to observe the same planewith another imaging scale, the invention provides that:

-   -   the current field diaphragm 13 is exchanged for a field        diaphragm 13 whose diaphragm opening is adapted to this second        objective, or    -   the current aperture diaphragm 14 is exchanged for an aperture        diaphragm 14 whose diaphragm opening is adapted to this        substituted objective, or    -   both diaphragms 13, 14 are exchanged at the same time to adapt        the cross section and the length 1 of the light sheet to the        substituted objective and, therefore, to make possible an        efficient observation of the specimen 1 in the selected plane.

The thickness d and length 1 of the light sheet 3 cannot be adjustedindependently from one another because, as was already stated, the depthof focus range depends upon the inverse square of the numerical apertureof the focused light bundle when the light sheet 3 is generatedanamorphotically by the aspherical optical element 7.

It is conceivable to change the diaphragms 13, 14 either manually orautomatically. In the latter case, the diaphragms 13, 14 are arranged,for example, on changer wheels coupled with the drives and the drivesare coupled with a control unit from which actuating commands are issuedwhich are generated depending on the characteristics of the substitutedobjective.

FIG. 2 shows the first construction of the arrangement according to theinvention in a variant in which zoom optics, which in this instancecomprise lens groups 15, 16 and 17, for example, are provided forinfluencing the geometry of the light sheet 3 instead of the Helddiaphragm and aperture diaphragm.

For the sake of clarity, the reference numbers used in FIG. 2 are thesame as those used in FIG. 1 for identical optical components. As inFIG. 1, the Y-Z plane is shown in FIG. 2a and the X-Z plane is shown inFIG. 2b.

To reshape the light bundle 5 into a light sheet 3, an asphericalelement 7 similar to that shown in FIG. 1 is again provided in thevariant according to FIG. 2, a lens group 8, preferably, an achromat,being arranged downstream of the aspherical element 7 in order torealize a field diaphragm plane. A light sheet 3 with a homogenizedradiation intensity in cross section is formed in the field diaphragmplane. The field diaphragm plane is located, for example, directly infront of a lens 18. The light sheet 3 is imaged in the object plane, andaccordingly in the specimen 1, by means of a lens 19 and the lens groups15, 16 and 17 of the zoom optics.

Influencing of the geometry of the light sheet 3 with the zoom optics iscarried out in that the focal length of the zoom is varied, for example,by means of axial displacement of the lens groups 15 and 17. The lenses18 to 21 ensure the correct pupil position within the zoom optics andguarantee a substantially collimated beam path in the field diaphragmplane in the Y-Z section. In this constructional variant, the lenses 20and 21 are constructed as cylindrical lenses.

FIG. 3 shows a modified variant of the first construction of thearrangement according to the invention which was mentioned above withreference to FIG. 1 and FIG. 2. The identical reference numbers areagain used to designate the same components.

Only the section in the Y-Z plane is shown; the X-Z plane is omittedsince it follows analogously from FIG. 1 and FIG. 2.

In contrast to and in addition to the variants according to FIG. 1 andFIG. 2, a device for reducing the formation of shadows in the specimen 1is provided in the variant according to FIG. 3. By reducing theformation of shadows, shadows are prevented on the specimen substancethat lies behind specimen particles not transparent to the illuminationlight in the direction of the illumination beam within the observedspecimen plane.

In this regard, it can be seen from FIG. 3 that after passing the fielddiaphragm 13 and the lens group 10 the light sheet 3 strikes a wobbleplate 22 which is located in a pupil plane of the illumination beam paththat is shaped to form the light sheet 3. Because of the oscillatingmovements of the wobble plate, the direction of the beam components ofthe light sheet 3 is influenced in such a way that they strike thespecimen substance successively in time in alternating directions and atdifferent angles so that non-transparent specimen substances areilluminated from behind and shadows caused by these specimen substanceswithin the illuminated specimen plane are prevented or at leastsubstantially reduced.

FIG. 4 shows another variant for reducing shadows relating to the firstconstruction of the arrangement according to the invention which wasdescribed above with reference to FIGS. 1 to 3. In this case, anoscillating mirror 23 is positioned in a plane conjugate to the fielddiaphragm plane 9. Because of its oscillating motion, the oscillatingmirror 23, similar to the wobble plate in FIG. 3, causes the directionof the beam components of the light sheet 3 to be influenced in such away that they strike the specimen substance in different directions.

In a second construction of the arrangement according to the inventionshown in FIG. 5, a light source which radiates a bundle of spatiallypartially coherent light is required. Two cylindrical-lens arrays 24 and25 are arranged in the path of the light coming from this light source(not shown in the drawing). Together with collimating optics 37, thesetwo cylindrical-lens arrays 24 and 25 form a honeycomb condenser whichacts in the Y-Z plane and through which an homogenization of theradiation intensity is achieved in this Y-Z plane. Only collimatingoptics 37 which generate the thickness d of the light sheet in thespecimen plane acts in the X-Z plane. The Y-Z plane is again shown inFIG. 5a and the X-Z plane is shown in FIG. 5b.

The spatially partially coherent light required for the arrangementaccording to the invention shown in FIG. 5 can be generated, forexample, as in FIG. 6.

In this case, a broadband laser 26 is provided. A temporally partiallycoherent beam proceeds from this broadband laser 26 and is directed to agrating 27. The grating 27 acts as a dispersive optical element so thatthe spatial coherence of the light reflected by the grating 27 isreduced. This light strikes the cylindrical-leas arrays 24 and 25 as isshown in FIG. 5. Alternatively, dispersive optical elements in the formof prisms or stepped mirrors can also be used instead of the grating 27.

FIG. 7 shows a third construction of the arrangement according to theinvention for generating a light sheet 3. In this case, the light sourcecomprises an array of individual laser light sources 28. Each lightsource generates a complete light sheet 3 by means of the cylindricallens 29 and the lens group 12. The spatial arrangement of the laserlight sources 28 influences the direction of individual radiationcomponents in such a way that shadows due to non-transparent specimensubstances within the illuminated specimen plane are prevented orsubstantially reduced.

It is also possible to use a GRIN lens instead of the cylindrical lens29.

FIG. 8 shows a fourth construction of the arrangement according to theinvention. In this case, a light bundle 5 coming from a coherent lightsource and exiting from a light-conducting fiber (not shown) is directedto a micro-optical element 30 which provides for the transformation ofthe light bundle 5 into the shape of a light sheet 3. This micro-opticalelement 30 can be, for example, micro-optics provided with opticallyactive free-form surfaces, or it can be constructed in the form of aGRIN lens.

Aside from transforming the light bundle 5 into the shape of the lightsheet 3, a micro-optical element 30 of the kind mentioned above alsoachieves a homogenizing action in the Y-Z plane (shown in FIG. 8a) and afocusing action in the orthogonal plane X-Z (shown in FIG. 8b).

Another, fifth construction of the arrangement according to theinvention is shown in FIG. 9. Realization of this construction againrequires a light source from which a light bundle 5 of coherent lightproceeds. The light bundle 5 is directed to anamorphotic optics, forexample, in the form of a cylindrical telescope 31 which causes anindependent cross-sectional change in the light bundle 5 in the X-axisand Y-axis so that the light bundle obtains an elliptical cross section.An optical deflecting device, in this case, for example, in the form ofan oscillating mirror 33, is arranged further along the path. It is alsoconceivable to use a polygon scanner or a DMD instead of the oscillatingmirror 33 to generate a scanning movement.

The light bundle which is deflected in a scanning manner by theoscillating mirror 33 is directed into the specimen 1 through the lensgroup 12.

As a result of the scanning movement of the oscillating mirror 33, theelliptical light bundle that is focused in the specimen is locatedsuccessively in time at positions 34, 35 and 36. The lining up of therespective illuminated areas in the specimen 1 yields the desired lightsheet 3.

In this construction, shadows are prevented or reduced within theobserved specimen plane due to the radiating angle occurring whenfocusing. The beam angles can be adjusted through the characteristics ofthe anamorphotic optics 31.

While the foregoing description and drawings represent the presentinvention, it will be obvious to those skilled in the art that variouschanges may be made therein without departing from the true spirit andscope of the present invention.

REFERENCE NUMBERS

-   1 specimen-   2 gel-   3 light sheet-   4 light-conducting fiber-   5 light bundle-   6 collimator-   7 aspherical optical element-   8 lens group-   9 field diaphragm plane-   10 lens group-   11 aperture diaphragm plane-   12 lens group-   13 field diaphragm-   14 aperture diaphragm-   15, 16, 17 lens groups-   18, 19, 20, 21 lenses-   22 wobble plate-   23 oscillating mirror-   24 cylindrical-lens array-   25 cylindrical-lens ray-   26 broadband laser-   27 grating-   28 laser light sources-   29 cylindrical lens-   30 micro-optical element-   31 cylindrical-lens telescope-   32 light bundle with elliptical cross section-   33 oscillating mirror-   34, 35, 36 positions-   37 collimating optics

The invention claimed is:
 1. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein theaspherical optical element is in the form of a Powell lens; and whereinthe light source emits a light bundle of coherent light, in whose pathare provided: a collimator; an aspherical optical element; and a lens orlens group for realizing a field diaphragm plane; and a lens or lensgroup for realizing an aperture diaphragm plane.
 2. An opticalarrangement for generating a light sheet, particularly for illuminatinga three-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: a light source for emitting a light bundle; opticalelements for transforming said light bundle into the shape of a lightsheet; means for varying the cross section; means for varying thelength; and/or means for influencing the direction in which beamcomponents extending within the light sheet are directed to the specimensubstance; wherein a plurality of field diaphragms which can beexchanged with one another in the field diaphragm plane and which havedifferent diaphragm openings are provided as means for varying the crosssection of the light sheet; and wherein the light source emits a lightbundle of coherent light, in whose path are provided: a collimator; anaspherical optical element; and a lens or lens group for realizing afield diaphragm plane; and a lens or lens group for realizing anaperture diaphragm plane.
 3. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein a pluralityof aperture diaphragms which can be exchanged with one another in theaperture diaphragm plane and which have different diaphragm openings areprovided as means for varying the length of the light sheet; and whereinthe light source emits a light bundle of coherent light in whose pathare provided: a collimator; an aspherical optical element; and a lens orlens group for realizing a field diaphragm plane; and a lens or lensgroup for realizing an aperture diaphragm plane.
 4. An opticalarrangement for generating a light sheet, particularly for illuminatinga three-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: a light source for emitting a light bundle; opticalelements for transforming said light bundle into the shape of a lightsheet; means for varying the cross section; means for varying thelength; and/or means for influencing the direction in which beamcomponents extending within the light sheet are directed to the specimensubstance; wherein a wobble plate is provided for influencing thedirection of beam components; and wherein the light source emits a lightbundle of coherent light, in whose path are provided: a collimator; anaspherical optical element; and a lens or lens group for realizing afield diaphragm plane; and a lens or lens group for realizing anaperture diaphragm plane.
 5. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein anoscillating mirror or a polygon scanner is provided for influencing thedirection of beam components; and wherein the light source emits a lightbundle of coherent light, in whose path are provided: a collimator; anaspherical optical element; and a lens or lens group for realizing afield diaphragm plane; and a lens or lens group for realizing anaperture diaphragm plane.
 6. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein the lightsource emits a light bundle of spatially partially coherent light, and acylindrical honeycomb condenser, preferably comprising twoone-dimensional cylindrical-lens arrays and collimating optics, isarranged in the path of this light bundle, wherein the lateral coherencelength is less than the period of the cylindrical-lens arrays.
 7. Theoptical arrangement according to claim 6; wherein a temporally partiallycoherent light source, particularly in the form of a broadband laser, isprovided for generating the spatially partially coherent light, adispersive optical element.
 8. The optical arrangement according toclaim 7; wherein the dispersive optical element is in the form of agrating and the coherent light source is in the form of a broadbandlaser.
 9. An optical arrangement for generating light sheet,particularly for illuminating a three-dimensional specimen in selectiveplane illumination microscopy (SPIM), comprising: a light source foremitting a light bundle; optical elements for transforming said lightbundle into the shape of a light sheet; means for varying the crosssection; means for varying the length; and/or means for influencing thedirection in which beam components extending within the light sheet aredirected to the specimen substance; wherein the light source comprisesan array of individual laser light sources, and a cylindrical lens or aGRIN lens is arranged downstream of this array.
 10. An opticalarrangement for generating a light sheet, particularly for illuminatinga three-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: a light source for emitting a light bundle; opticalelements for transforming said light bundle into the shape of a lightsheet; means for varying the cross section; means for varying thelength; and/or means for influencing the direction in which beamcomponents extending within the light sheet are directed to the specimensubstance; wherein a micro-optical element is provided for transformingthe light bundle into the shape of a light sheet and has a homogenizingeffect in one cross-sectional axis and, at the same time, a focusingeffect in the orthogonal cross-sectional axis.
 11. The opticalarrangement according to claim 10; wherein the micro-optical element hasoptically active free-form surfaces or in the form of a GRIN lens. 12.An optical arrangement for generating a light sheet, particularly forilluminating a three-dimensional specimen in selective planeillumination microscopy (SPIM), comprising: a light source for emittinga light bundle; optical elements for transforming said light bundle intothe shape of a light sheet; means for varying the cross section; meansfor varying the length; and/or means for influencing the direction inwhich beam components extending within the light sheet are directed tothe specimen substance; wherein the light source emits a light bundle ofcoherent light, within whose path are provided: anamorphotic optics forgenerating a light bundle with an elliptic cross section; an opticaldeflection device downstream of the latter for generating a scanningmovement of this light bundle; and a focusing lens or lens group throughwhich the scanned light bundle is directed to the specimen.
 13. Theoptical arrangement according to claim 12; wherein the anamorphoticoptics is in the form of a cylindrical-lens telescope and the opticaldeflection device is preferably in the form of an oscillating mirror.14. An optical arrangement for generating a light sheet for illuminatinga three-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: means for forming a light sheet from light; andlight sheet influencing means for influencing the direction in whichbeam components extending within the light sheet are directed to thespecimen substance such that the beam components strike the specimensubstance successively in time in alternating directions such thatshadows caused by non-transparent specimen substances within theilluminated plane are at least substantially reduced or prevented. 15.The optical arrangement according to claim 14 wherein the light sheetinfluencing means comprises: an optical deflection device, wherein theoptical deflection device is positioned such that when the light sheetstrikes the optical deflection device, oscillating movements of theoptical deflection device influence the direction of beam components tostrike the specimen substance successively in time in alternatingdirections.
 16. The optical arrangement according to claim 15 whereinthe optical deflection device comprises: a wobble plate.
 17. The opticalarrangement according to claim 16, comprising: the wobble plate beinglocated in a pupil plane of the illumination beam path of the lightsheet and positioned such that when the light sheet strikes the wobbleplate, the oscillating movements of the wobble plate influence thedirection of beam components to strike the specimen substancesuccessively in time in alternating directions and at alternatingangles.
 18. The optical arrangement according to claim 15 wherein theoptical deflection device comprises: an oscillating mirror or polygonscanner.
 19. The optical arrangement according to claim 18 comprising:the oscillating mirror or polygon scanner being positioned in a planeconjugate to a field diaphragm plane.
 20. The optical arrangementaccording to claim 14 comprising: a light source for emitting a lightbundle; and wherein the means for forming a light sheet from lightcomprise optical elements for transforming the light bundle into theshape of the light sheet.
 21. A method for generating a light sheet forilluminating a three-dimensional specimen in selective planeillumination microscopy (SPIM), comprising: forming a light sheet fromlight; and deflecting the light sheet to influence the direction inwhich beam components extending within the light sheet are directed tothe specimen substance such that the beam components strike the specimensubstance successively in time in alternating directions such thatshadows caused by non-transparent specimen substances within theilluminated plane are at least substantially reduced or prevented. 22.The method according to claim 21 wherein method comprises: deflectingthe light sheet using an optical deflection device positioned in anoptical arrangement such that when the light sheet strikes the opticaldeflection device, oscillating movements of the optical deflectiondevice influence the direction of beam components to strike the specimensubstance successively in time in alternating directions.
 23. The methodaccording to claim 22 wherein the method comprises: deflecting the lightsheet using a wobble plate positioned in a pupil plane of theillumination beam path of the light sheet, wherein the wobble plate ispositioned such that that when the light sheet strikes the wobble plate,the oscillating movements of the wobble plate influence the direction ofbeam components to strike the specimen substance successively in time inalternating directions and at alternating angles.
 24. The methodaccording to claim 22 wherein the method comprises: deflecting the lightsheet using an oscillating mirror or polygon scanner, the oscillatingmirror or polygon scanner being positioned in a plane conjugate to afield diaphragm plane.
 25. The method according to claim 21 wherein themethod comprises: emitting a light bundle from a light source; andwherein the forming the light sheet from light comprises transformingthe light bundle into the shape of the light sheet with opticalelements.
 26. An optical arrangement for generating a light sheet,particularly for illuminating a three-dimensional specimen in selectiveplane illumination microscopy (SPIM), comprising: a light source foremitting a light bundle; optical elements for transforming said lightbundle into the shape of a light sheet; means for varying the crosssection; means for varying the length; and/or means for influencing thedirection in which beam components extending within the light sheet aredirected to the specimen substance; wherein the aspherical opticalelement is in the form of a Powell lens; and wherein the light sourceemits a light bundle of coherent light, in whose path are provided: acollimator; an aspherical optical element; and a lens or lens group forrealizing a field diaphragm plane; and a lens or lens group forrealizing an aperture diaphragm plane.
 27. An optical arrangement forgenerating a light sheet, particularly for illuminating athree-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: a light source for emitting a light bundle; opticalelements for transforming said light bundle into the shape of a lightsheet; means for varying the cross section; means for varying thelength; and/or means for influencing the direction in which beamcomponents extending within the light sheet are directed to the specimensubstance; wherein a plurality of field diaphragms which can beexchanged with one another in the field diaphragm plane and which havedifferent diaphragm openings are provided as means for varying the crosssection of the light sheet; and wherein the light source emits a lightbundle of coherent light, in whose path are provided: a collimator; anaspherical optical element; and a lens or lens group for realizing afield diaphragm plane; and a lens or lens group for realizing anaperture diaphragm plane.
 28. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein a pluralityof aperture diaphragms which can be exchanged with one another in theaperture diaphragm plane and which have different diaphragm openings areprovided as means for varying the length of the light sheet; and whereinthe light source emits a light bundle of coherent light in whose pathare provided: a collimator; an aspherical optical element; and a lens orlens group for realizing a field diaphragm plane; and a lens or lensgroup for realizing an aperture diaphragm plane.
 29. An opticalarrangement for generating a light sheet, particularly for illuminatinga three-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: a light source for emitting a light bundle; opticalelements for transforming said light bundle into the shape of a lightsheet; means for varying the cross section; means for varying thelength; and/or means for influencing the direction in which beamcomponents extending within the light sheet are directed to the specimensubstance; wherein a wobble plate is provided for influencing thedirection of beam components; and wherein the light source emits a lightbundle of coherent light, in whose path are provided: a collimator; anaspherical optical element; and a lens or lens group for realizing afield diaphragm plane; and a lens or lens group for realizing anaperture diaphragm plane.
 30. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein anoscillating mirror or a polygon scanner is provided for influencing thedirection of beam components; and wherein the light source emits a lightbundle of coherent light, in whose path are provided: a collimator; anaspherical optical element; and a lens or lens group for realizing afield diaphragm plane; and a lens or lens group for realizing anaperture diaphragm plane.
 31. An optical arrangement for generating alight sheet, particularly for illuminating a three-dimensional specimenin selective plane illumination microscopy (SPIM), comprising: a lightsource for emitting a light bundle; optical elements for transformingsaid light bundle into the shape of a light sheet; means for varying thecross section; means for varying the length; and/or means forinfluencing the direction in which beam components extending within thelight sheet are directed to the specimen substance; wherein the lightsource emits a light bundle of spatially partially coherent light, and acylindrical honeycomb condenser, preferably comprising twoone-dimensional cylindrical-lens arrays and collimating optics, isarranged in the path of this light bundle, wherein the lateral coherencelength is less than the period of the cylindrical-lens arrays.
 32. Theoptical arrangement according to claim 31; wherein a temporallypartially coherent light source, particularly in the form of a broadbandlaser, is provided for generating the spatially partially coherentlight, a dispersive optical element.
 33. The optical arrangementaccording to claim 32; wherein the dispersive optical element is in theform of a grating and the coherent light source is in the form of abroadband laser.
 34. An optical arrangement for generating light sheet,particularly for illuminating a three-dimensional specimen in selectiveplane illumination microscopy (SPIM), comprising: a light source foremitting a light bundle; optical elements for transforming said lightbundle into the shape of a light sheet; means for varying the crosssection; means for varying the length; and/or means for influencing thedirection in which beam components extending within the light sheet aredirected to the specimen substance; wherein the light source comprisesan array of individual laser light sources, and a cylindrical lens or aGRIN lens is arranged downstream of this array.
 35. An opticalarrangement for generating a light sheet, particularly for illuminatinga three-dimensional specimen in selective plane illumination microscopy(SPIM), comprising: a light source for emitting a light bundle; opticalelements for transforming said light bundle into the shape of a lightsheet; means for varying the cross section; means for varying thelength; and/or means for influencing the direction in which beamcomponents extending within the light sheet are directed to the specimensubstance; wherein a micro-optical element is provided for transformingthe light bundle into the shape of a light sheet and has a homogenizingeffect in one cross-sectional axis and, at the same time, a focusingeffect in the orthogonal cross-sectional axis.
 36. The opticalarrangement according to claim 35; wherein the micro-optical element hasoptically active free-form surfaces or in the form of a GRIN lens. 37.An optical arrangement for generating a light sheet, particularly forilluminating a three-dimensional specimen in selective planeillumination microscopy (SPIM), comprising: a light source for emittinga light bundle; optical elements for transforming said light bundle intothe shape of a light sheet; means for varying the cross section; meansfor varying the length; and/or means for influencing the direction inwhich beam components extending within the light sheet are directed tothe specimen substance; wherein the light source emits a light bundle ofcoherent light, within whose path are provided: anamorphotic optics forgenerating a light bundle with an elliptic cross section; an opticaldeflection device downstream of the latter for generating a scanningmovement of this light bundle; and a focusing lens or lens group throughwhich the scanned light bundle is directed to the specimen.
 38. Theoptical arrangement according to claim 37; wherein the anamorphoticoptics is in the form of a cylindrical-lens telescope and the opticaldeflection device is preferably in the form of an oscillating mirror.